The signaling molecules that regulate embryonic (primitive) erythropoiesis are not known and their discovery could provide new candidate targets for treating hematopoietic disorders and disease. One family of signaling molecules that is clearly implicated are the bone morphogenetic proteins (BMPs) of the TGF-Beta super-family. Smad proteins are key mediators of TGF-Beta-like signaling. However, BMPs and Smads function in many diverse processes during development and there is a current lack of understanding for how they regulate specific genetic programs. The hypothesis to be tested is that regulation of primitive erythropoiesis is controlled by specific Smad proteins and cell-restricted Smad-interacting proteins. The primitive erythroid lineage has been a challenge to study, since it is transient and mutations in regulatory genes can result in early embryonic lethality. Therefore, innovative systems are needed. In this proposal, three complementary systems are exploited to discover conserved mechanisms controlling embryonic erythropoiesis. The Xenopus system provides advantages for deregulating specific genes by ectopic expression and antisense approaches, avian embryos provide abundant pure primary erythroid cells for biochemistry, while zebrafish embryos can be used for genetic manipulation in the primitive erythroid lineage. The goal of this proposal is to identify proteins that control development of the first erythroid cells in response to BMP signaling. Aims are proposed to investigate the role of specific Smads and identify relevant interacting proteins with significance for primitive hematopoiesis. First, factors that mediate BMP-dependent activation of the GATA-2 gene will be characterized using embryonic and erythroid cell extracts. Candidate screens and more general biochemical approaches will be used to define the Smads and cell-restricted proteins that interact with a defined response element. Second, the ability of specific Smads to regulate primitive erythropoiesis will be tested. Smad signaling will be deregulated in several contexts, including the use of antisense morpholinos in frogs, and targeted transgenics in zebrafish. Third, novel regulatory factors that interact with Smads will be identified and tested for relevance to primitive erythropoiesis, using biochemical and genetic approaches. It is anticipated that results from these proposed experiments will provide new information regarding the specificity and regulation of primitive erythropoiesis.